Pyramiding of transcription factor, PgHSF4, and stress-responsive genes of p68, Pg47, and PsAKR1 impart multiple abiotic stress tolerance in rice (Oryza sativa L.).

Abiotic stresses such as drought, salinity, and heat stress significantly affect rice crop growth and production. Under uncertain climatic conditions, the concurrent multiple abiotic stresses at different stages of rice production became a major challenge for agriculture. Hence, improving rice's multiple abiotic stress tolerance is essential to overcome unprecedented challenges under adverse environmental conditions. A significant challenge for rice breeding programs in improving abiotic stress tolerance involves multiple traits and their complexity. Multiple traits must be targeted to improve multiple stress tolerance in rice and uncover the mechanisms. With this hypothesis, in the present study gene stacking approach is used to integrate multiple traits involved in stress tolerance. The multigene transgenics co-expressing Pennisetum glaucum 47 (Pg47), Pea 68 (p68), Pennisetum glaucum Heat Shock Factor 4(PgHSF4), and Pseudomonas Aldo Keto Reductase 1 (PsAKR1) genes in the rice genotype (AC39020) were developed using the in-planta transformation method. The promising transgenic lines maintained higher yields under semi-irrigated aerobic cultivation (moisture stress). These 15 promising transgenic rice seedlings showed improved shoot and root growth traits under salinity, accelerating aging, temperature, and oxidative stress. They showed better physiological characteristics, such as chlorophyll content, membrane stability, and lower accumulation of reactive oxygen species, under multiple abiotic stresses than wild-type. Enhanced expression of transgenes and other stress-responsive downstream genes such as HSP70, SOD, APX, SOS, PP2C, and P5CS in transgenic lines suggest the possible molecular mechanism for imparting the abiotic stress tolerance. This study proved that multiple genes stacking as a novel strategy induce several mechanisms and responsible traits to overcome multiple abiotic stresses. This multigene combination can potentially improve tolerance to multiple abiotic stress conditions and pave the way for developing climate-resilient crops.

Identification of Putative Molecular Markers Associated with Root Traits in Coffea canephora Pierre ex Froehner.

Coffea canephora exhibit poor root system and are very sensitive to drought stress that affects growth and production. Deeper root system has been largely empirical as better avoidance to soil water limitation in drought condition. The present study aimed to identify molecular markers linked to high root types in Coffea canephora using molecular markers. Contrasting parents, L1 valley with low root and S.3334 with high root type, were crossed, and 134 F1 individuals were phenotyped for root and associated physiological traits (29 traits) and genotyped with 41 of the 320 RAPD and 9 of the 55 SSR polymorphic primers. Single marker analysis was deployed for detecting the association of markers linked to root associated traits by SAS software. There were 13 putative RAPD markers associated with root traits such as root length, secondary roots, root dry weight, and root to shoot ratio, in which root length associated marker OPS1850 showed high phenotypic variance of 6.86%. Two microsatellite markers linked to root length (CPCM13400) and root to shoot ratio (CM211300). Besides, 25 markers were associated with more than one trait and few of the markers were associated with positively related physiological traits and can be used in marker assisted trait selection.

Determination of IAA in brassinolide treated coleoptiles of wheat by a modified indirect ELISA with polyclonal antibodies.

An indirect enzyme immunoassay for detection of as little as 10-50 pmole IAA is described for the first time. The assay is based on the development of highly specific polyclonal antibodies against the carboxyl site of IAA. The binding specificity is nearly as high as the radioimmunoassay and the titre of the specific antibody was also remarkably high (1:40,000 of the primary antibody). Such an easy, rapid, specific and highly sensitive assay would be extremely useful in gaining more information on the mode of action of phytohormones, and their effects on physiological processes.

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress.

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.

Overexpression of a pea DNA helicase (PDH45) in peanut (Arachis hypogaea L.) confers improvement of cellular level tolerance and productivity under drought stress.

Peanut, a major edible oil seed crop globally is predominantly grown under rainfed conditions and suffers yield losses due to drought. Development of drought-tolerant varieties through transgenic technology is a valid approach. Besides superior water relation traits like water mining, intrinsic cellular level tolerance mechanisms are important to sustain the growth under stress. To achieve this objective, the focus of this study was to pyramid drought adaptive traits by overexpressing a stress responsive helicase, PDH45 in the background of a genotype with superior water relations. PCR, Southern, and RT-PCR analyses confirmed stable integration and expression of the PDH45 gene in peanut transgenics. At the end of T3 generation, eight transgenic events were identified as promising based on stress tolerance and improvement in productivity. Several transgenic lines showed stay-green phenotype and increased chlorophyll stability under stress and reduced chlorophyll retardation under etherel-induced simulated stress conditions. Stress-induced root growth was also substantially higher in the case of transformants. This was reflected in increased WUE (low Δ¹³C) and improved growth rates and productivity. The transgenics showed 17.2 and 26.75 % increase in yield under non-stress and stress conditions over wild type ascertaining the feasibility of trait pyramiding strategy for the development of drought-tolerant peanut.

Oxygen isotope enrichment (delta18O) as a measure of time-averaged transpiration rate.

Experimental evidence is presented to show that the 18O enrichment in the leaf biomass and the mean (time-averaged) transpiration rate are positively correlated in groundnut and rice genotypes. The relationship between oxygen isotope enrichment and stomatal conductance (g(s)) was determined by altering g(s) through ABA and subsequently using contrasting genotypes of cowpea and groundnut. The Peclet model for the 18O enrichment of leaf water relative to the source water is able to predict the mean observed values well, while it cannot reproduce the full range of measured isotopic values. Further, it fails to explain the observed positive correlation between transpiration rate and 18O enrichment in leaf biomass. Transpiration rate is influenced by the prevailing environmental conditions besides the intrinsic genetic variability. As all the genotypes of both species experienced similar environmental conditions, the differences in transpiration rate could mostly be dependent on intrinsic g(s). Therefore, it appears that the delta18O of leaf biomass can be used as an effective surrogate for mean transpiration rate. Further, at a given vapour pressure difference, delta18O can serve as a measure of stomatal conductance as well.

A novel colorimetric assay and activity staining for cytokinin oxidase based on the copper amine oxidase property of the enzyme.

A rapid and sensitive colorimetric assay for cytokinin oxidase (CKO) was developed based on the copper amine oxidase property of the enzyme from cucumber cotyledons. The assay involved oxidation of isopentenyl adenine by CKO in the presence of phenazine methosulphate as an electron acceptor, resulting in reduction of nitroblue tetrazolium to formazon. The formation of formazon was linear with time for at least 20 min and could be followed spectrophotometrically at A490. CKO could also be specifically stained on polyacrylamide gels using the same principle.